Hematocrit and the risk of coronary heart disease mortality in the TAMRISK study, a 28-year follow-up.

OBJECTIVE: To evaluate whether hematocrit (HCT) is associated with coronary heart disease (CHD) mortality in men over 55 years of age in Finland. METHODS: Health survey data were recorded in 1980 from 670 men, aged 55 years. The causes of deaths during a 28-year follow-up were obtained from official records. Statistical comparisons were done by Cox proportional hazard regression model after dividing the men into two groups, one with HCT<50% and the other, HCT> or =50%. RESULTS: There were altogether 412 deaths of all causes, including 140 from CHD. In men having HCT<50%, the crude CHD mortality rate per 10,000 population was 2203, while in men with HCT> or =50%, the corresponding figure was 4255. Men with HCT> or =50% were 2.4 times (95% CI 1.6-3.5) more likely to die from CHD than were men with HCT<50%. After adjusting for established coronary risk factors, the increased risk remained 1.8-fold (95 % CI, 1.1-2.7). CONCLUSIONS: Borderline polycythemia was associated with increased CHD mortality. The cut-off value in our study was > or =50%, proposing that for men over 55 years of age such HCT levels might be an additional risk factor.

Long-term survival after ischemic stroke in postmenopausal women is affected by an interaction between smoking and genetic variation in nitric oxide synthases.

BACKGROUND: We aimed to study whether variations in vasoregulatory endothelial nitric oxide synthase (eNOS 4a/b) and tissue-injury-associated inducible nitric oxide synthase (iNOS R5/4) genes and smoking might explain gender differences in long-term survival after stroke. METHODS: A total of 486 consecutive acute stroke patients, subjected to MRI, were followed up for a mean of 7.6 years. The eNOS 4a/b (n = 300) and iNOS R5/4 (n = 310) genotypes were determined by PCR. Of these patients, 213/300 (71.0%; eNOS 4a/b) and 223/310 (71.9%; iNOS R5/4) had died. RESULTS: Despite the fact that women were older than men (72.3 vs. 69.5 years, p = 0.001) at recruitment, poor long-term survival was not sex-related, but instead predicted by age (p < 0.0001), cardiac failure (p = 0.004), smoking (p = 0.017), diabetes (p = 0.049), and variation in the eNOS gene locus (p = 0.033). Smoking and variations in both eNOS [hazard ratio (HR) = 1.53, p = 0.011] and iNOS loci (HR = 1.52, p = 0.073) were found to impact upon poor survival. We found a strong interaction between smoking, female sex, and the iNOS R5/4 genotype with the risk of death (HR = 3.23, CI = 1.51-6.90, p = 0.002). Compared with nonsmoking noncarriers, postmenopausal women who had been smokers and carried either the rare iNOS R5 allele (17.1%; HR = 4.23, CI = 1.84-9.75, p = 0.001) or the common eNOS 4b allele (71%; HR = 3.14, CI = 1.49-6.62, p = 0.003) were at a higher risk of death during the follow-up. These interactions were independent of each other, and were not found among men. CONCLUSIONS: The interaction between smoking and genetic variants of eNOS and iNOS predicts survival after stroke, especially among postmenopausal women.

Estrogen receptor-1 genotype is related to coronary intima thickness in young to middle-aged women.

OBJECTIVE: The incidence of coronary disease in premenopausal women is about one-half that in men of similar age. The estrogen receptor-1 (ESR1, c.454-397T>C) CC variant genotype is associated with the severity of coronary artery disease (CAD) and an increased risk of myocardial infarction in men. The purpose of the present study was to investigate whether this ESR1 CC variant also disposes to atherosclerosis in women in terms of increased total coronary artery intima thickness. MATERIAL AND METHODS: A total of 125 forensic autopsy cases of women aged 15 to 49 years were investigated. The thickness of the coronary intima, which reflects the severity of atherosclerosis, was measured by computerized image analysis. The ESR1 c.454-397T>C genotype was determined by polymerase chain reaction (PCR). RESULTS: The mean intima thicknesses in the three genotype groups were 428+/-298 microm (TT), 494+/-371 microm (CT) and 636+/-436 microm (CC). We found that, on average, women with the CC genotype had a thicker coronary intima compared with that of women with the TT genotype, even after adjusting for age and body mass index (BMI) (p = 0.030). The intermediate group (TC) did not significantly differ from either the CC or the TT genotype group in this respect. CONCLUSION: Our results point to the importance of ESR1 genotype in relation to cardiovascular disease susceptibility.

Serum fatty acids in postinfarction middle-aged men.

There is a multitude of data showing that coronary heart disease is affected by the quality of dietary fat. The fatty acid composition of serum lipids has been shown to reflect that of the diet. It is likely that, after myocardial infarction, both the health-care professionals and the patients themselves pay more attention to dietary guidelines. In order to assess the correctness of this assumption, we compared the composition of serum fatty acids in 40 male subjects with a history of myocardial infarction (MI) with that of 40 age-matched controls, both from the FINRISK study. The percentage composition of fatty acids of total serum lipids was analysed by gas chromatography. In comparison with the control group, the MI group had higher body mass index (BMI), a higher prevalence of diabetes, higher level of serum triglycerides and a lower level of serum high-density lipoprotein (HDL) cholesterol, all indicators of the metabolic syndrome. The MI group had higher proportions of serum palmitic (16:0) and oleic acids (18:1), and a lower proportion of linoleic (18:2 n-6) acid than the control group. The metabolic syndrome is accompanied by an elevated level of serum insulin, which is known to enhance the synthesis of saturated and monounsaturated fatty acids, such as 16:0 and 18:1, and to stimulate the activity delta-6 desaturase, decreasing the concentration of linoleic acid. Our results suggest that the observed serum fatty acid composition in subjects with coronary heart disease is dependent on metabolic factors in addition to dietary fatty acid composition.

A functional variant of the iNOS gene flanking region is associated with LAD coronary artery disease: an autopsy study.

BACKGROUND: Recent studies using reporter gene constructs have indicated significant differences in the promoter activity of inducible nitric oxide synthase (iNOS) gene variants. Although the exact role of iNOS in atherogenesis is unclear, it is possible that this variation site may influence the extent of coronary artery disease (CAD). METHODS: We amplified these (AAAT) repeat variants from the NOS2A gene (denoted iNOS R4 and iNOS R5) from 325 Finnish men included in the Helsinki Sudden Death Study, and studied their association with indices of stenosis and atherosclerosis of the left anterior descending artery (LAD), right coronary artery (RCA) and left circumflex artery (LCX). In order to understand the effect of iNOS genotype on different stages of CAD, our study population was divided into age groups. RESULTS: In the LAD, the progression of atherosclerosis seemed to be more pronounced in the 4/5 genotype carriers than in those with the 4/4 genotype when the different age groups were compared. More specifically, statistically significant differences between the genotypes were found in the subgroup of men aged > 55 years. In this group, men carrying the rare R4/5 genotype presented higher mean values of stenosis percentages (55% vs. 42%, P = 0.008), larger areas of fatty streaks (10.4% vs. 5.9%; P = 0.01) and complicated lesions (3.5% vs. 1.3%; P = 0.001) compared with the R4/4 carriers. No significant association of iNOS genotypes with stenosis and atherosclerosis of RCA and LCX was found. CONCLUSIONS: It appears unlikely the R4/5 genotype plays a major role in the pathogenesis of CAD, as it was not associated with stenosis and atherosclerosis in RCA and LCX. However this genotype may have some role in more pronounced CAD, as seen in the LAD.

Fibrinogen gene promoter -455 A allele as a risk factor for lacunar stroke.

BACKGROUND AND PURPOSE: Elevated fibrinogen levels are suggested to increase the risk of myocardial infarction and stroke. Carriers of the A allele of the fibrinogen -455G/A polymorphism have increased plasma fibrinogen levels. We studied the association of this polymorphism with stroke subtype in the Stroke Aging Memory (SAM) cohort. METHODS: The SAM cohort comprises 486 consecutive patients 55 to 85 years of age who, 3 months after ischemic stroke, completed a detailed stroke assessment. Stroke subtypes were examined with MRI. -455G/A genotype was determined by polymerase chain reaction. MRI and genotype data were available for the 299 patients who constitute the present study population. RESULTS: Genotype distributions were 64.9% (GG), 31.8% (GA), and 3.3% (AA). In a logistic regression model with age, sex, hypertension, diabetes, hypercholesterolemia, hypertriglyceridemia, myocardial infarction, arrhythmia, atrial fibrillation, peripheral arterial disease, and smoking as possible confounders, there was a significant association between A+ genotype and >or=3 lacunar infarcts (odds ratio [OR], 2.57; 95% CI, 1.23 to 5.36; P=0.01). Hypertensive patients carrying the A allele had increased risk (OR, 4.24; 95% CI, 1.29 to 13.99; P=0.02) for >or=3 lacunar infarcts. A similar increase in risk was observed among smokers with the A+ genotype (OR, 2.67; 95% CI, 0.92 to 7.77; P=0.07). CONCLUSIONS: Stroke patients carrying the A allele of the Bbeta-fibrinogen -455G/A polymorphism frequently presented with multiple lacunar infarcts. This association was stronger among hypertensives and smokers. These associations suggest that the A allele may predispose to atherothrombotic events in cerebrovascular circulation.

Association of neuropeptide y polymorphism with the occurrence of type 1 and type 2 alcoholism.

BACKGROUND: The susceptibility to alcoholism can be explained partially by genetic factors. Neuropeptide Y (NPY) has emerged as one potential factor contributing the development of alcoholism. A recent study indicated that the NPY gene variant producing a leucine-to-proline substitution (T to C at position 1128) was associated with 34% higher average alcohol consumption. METHODS: The subjects consisted of 122 alcoholics classified as type 1 and type 2 subtypes by psychiatric evaluation. A random sample of 59 social drinkers was used as a control group to compare the distribution of NPY genotypes with those of alcoholics. RESULTS: In a logistical regression model, there was a significantly lower frequency of the leucine(7)/proline(7) heterozygotes among well characterized type 2 alcoholics, compared with the controls (10.8 vs. 24.1%, p = 0.028). CONCLUSIONS: We speculate that the genetic polymorphism producing the proline(7) substitution of NPY might not predispose to alcoholism, but indeed retard the transition to alcoholism.

Association of apolipoprotein E polymorphism with outcome after aneurysmal subarachnoid hemorrhage: a preliminary study.

BACKGROUND AND PURPOSE: Variation in the outcome after aneurysmal subarachnoid hemorrhage (SAH) is not fully explained by known prognostic factors. APOE genotype is the most important genetic determinant of susceptibility to Alzheimer's disease, and it is also shown to be associated with the outcome after traumatic brain injury. We studied the association of apolipoprotein E polymorphism with the outcome after aneurysmal SAH. METHODS: A total of 160 consecutive patients were admitted after SAH to a neurosurgical unit. The clinical assessment after the SAH was performed with the Hunt and Hess grading scale. The severity of the bleeding as visualized on CT was assessed by Fisher's grading system. Outcome was assessed with the Glasgow Outcome SCALE: APOE genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: 126 patients had aneurysmatic SAH, and detailed information on outcome and APOE genotype was available for 108 patients (86%). Sixteen (40%) of 40 patients with APOE epsilon4 had an unfavorable outcome compared with 13 (19%) of 68 without the APOE epsilon4 allele (OR 2.8, 95% CI 1.18 to 6.77). Association was more significant after adjustment for age, rebleeding, clinical status on admission, and CT scan findings (OR 7.1, 95% CI 1.9 to 26.3; P=0.0035). CONCLUSIONS: Our findings show a significant genetic association of APOE polymorphism with outcome after spontaneous aneurysmal SAH. Genetic factors thus seem to explain a part of individual differences in the recovery of SAH.

Polymorphisms within the tumor necrosis factor locus and prevalence of coronary artery disease in middle-aged men.

Tumor necrosis factor (TNF) is an important cytokine in the inflammation process of atherosclerosis and is also involved in lipid metabolism. Two biallelic polymorphisms within TNF gene locus-TNFA at the position -308 in the promoter region of the TNF gene and TNFB in the first intron of the lymphotoxin-alpha (LT-alpha) have been reported to be associated with TNF production and with susceptibility to inflammatory diseases. We studied the association of these polymorphisms within the major histocompatibility complex (MHC) III region with coronary atherosclerosis and its manifestations. The autopsy series comprised 700 Caucasian Finnish men, aged 33-70 years (The Helsinki Sudden Death Study). Coronary stenosis and surface area of atherosclerotic changes (fatty streaks, fibrous plaques, complicated lesions and calcification) were measured and the presence of myocardial infarction and coronary thrombosis recorded. TNFA and TNFB genotypes were determined by the PCR-RFLP technique. The allele frequencies were TNFA1/TNFA2=0.88/0.12 and TNFB1/TNFB2=0.30/0.70. There was a strong linkage disequilibrium between the two polymorphisms. There were no differences in coronary stenosis and in the frequency of old or recent myocardial infarction or coronary thrombosis between men with different genotype status in either locus. Men with TNFA22 or TNFB11 genotype tended to have more fibrous lesions and calcification in their coronary arteries. TNFA and TNFB polymorphisms are unlikely to contribute to progression of atherosclerosis in a way clinically important.

Analysis of estrogen receptor dinucleotide polymorphism by capillary gel electrophoresis with a population genetic study in 180 Finns.

We developed a suitable method for analysing dinucleotide repeats found in the upstream of human alpha-estrogen receptor (ER) gene by applying capillary electrophoresis and automatic analysis. This method omits the gel-casting step as well as difficult handling of long polyacrylamide sequencing gels. Use of radioactive materials is also avoided. Using this method, the frequency distribution of ER alleles, determined in 180 Finnish individuals showed two peaks at 12 and 14 repeats (166 and 168 bp) and also at 22 and 24 repeats (184 and 186 bp). The overall distribution of alleles seemed to be similar to that found among Italian and Japanese populations.

Long-extension PCR to detect deleted mitochondrial DNA molecules is compromized by technical artefacts.

Long-extension PCR (LX-PCR), followed by Southern hybridization to probes for two different regions of the mitochondrial genome, was used to evaluate the presence of deleted mtDNA molecules in heart muscle samples from alcoholic cardiomyopathy patients compared with age-matched controls. Two different primer pairs capable of amplifying the entire genome, as well as a variety of other primer pairs predicted to amplify the genome in large, overlapping fragments, were tested. Products indicating the presence of a variety of subgenomic, deleted molecules were detected in variable amounts from patient and control myocardial samples alike. Most of these hybridized with a probe for the 16S/ND1 region, but not with a probe for the ND4/ND5 region that is commonly deleted. Dilution of a given template DNA in which deleted products were prominent resulted in the disappearance of the subgenomic bands in favour of the full-length, undeleted product. Therefore, the appearance and amount of such products is subject to template concentration or quality. The results indicate that the application of LX-PCR to the detection and quantitation of deleted mtDNAs is inherently unreliable, and findings using this technique should be treated with caution unless supported by an independent method.

Induction of chicken avidin and related mRNAs after bacterial infection.

Inflammation-mediated avidin gene expression in the chicken was studied using hybridization analysis, polymerase chain reaction (RT-PCR) and sequencing. The results indicate the presence of avidin mRNA in the oviduct and intestine after Escherichia coli infection. The mRNA for the inflammation-induced avidin was mainly encoded by the avidin gene, but the avidin-related genes, avr2 and avr3, were also transcribed at a low level in the oviduct and intestine, respectively. Because avidin is also induced in the chicken oviduct by progesterone, our results indicate a multifactorial regulation of avidin gene expression.

Inducibility of the avidin gene by progesterone is suppressed during estrogen-induced cytodifferentiation.

We have studied epithelial differentiation of the chick oviduct as induced by diethylstilbestrol (DES) and 17 beta-estradiol (E2). The proportion of goblet cells in the oviduct was slightly higher after E2 than after DES treatment. Also avidin induction by progesterone was stronger following DES than E2 priming. In the estrogen pretreated oviduct epithelium, avidin expression was induced by progesterone in the surface epithelial cells, protodifferentiated gland cells and tubular gland cells, but not in goblet cells. During prolonged estrogen treatment, however, the inducibility of avidin by progesterone ceased in tubular gland cells but not in surface epithelial cells. The estrogen action on the expression of avidin could be explained by estrogen-induced terminal differentiation of the epithelial gland cells or by a direct effect of estrogen on the progesterone action, for instance interaction of estrogen receptor and progesterone receptor in the regulation of transcription.

Induction of avidin messenger ribonucleic acid in the chick oviduct by progesterone and other steroids.

Avidin gene expression was analyzed using an avidin immunoassay and RNA hybridization analysis. To ascertain whether the induction of the avidin gene by progesterone remains specific also during secondary restimulation with diethylstilbestrol, chicks were given different steroid hormones or hormone combinations. Progesterone-specific induction of avidin protein and messenger RNA (mRNA) was 15- to 30-fold over the control even after secondary restimulation with diethylstilbestrol. A functional difference between the progesterone response element and glucocorticoid response element was suggested, since dexamethasone alone did not induce avidin in vivo. In spite of progesterone specificity, a combination of progesterone with other steroids nevertheless generated a synergistic increase in the amount of avidin mRNA. This may indicate that binding of progesterone receptor to the progesterone response element may be important to alter the functional activity of other hormone response elements present on the avidin gene. The time response curve of the avidin mRNA induction by progesterone was also determined. Avidin mRNA was detectable 8 h after progesterone induction, and its amount was maximal after 16-24 h. This would indicate that the avidin gene belongs in the so-called late responder genes, which also include chicken ovalbumin, ovomucoid, and lysozyme genes.

Development of progestin-specific response in the chicken oviduct.

Avidin is a host acute defense protein induced by progestins and by inflammation caused by injurious factors such as microbes, viruses, toxic factors or tissue trauma. In the reproductive tract of egg-laying vertebrates avidin has evolved into a progestin-dependent secretory protein involved in anti-microbial action through its biotin avidity. For "progestin-dependent avidin" production, cellular differentiation by estrogen is necessary. In contrast, the expression of "progestin-independent or inflammation-induced avidin" does not require differentiation. Many cell types such as macrophages, heterophils and fibroblasts can produce avidin after non-specific cellular injuries. The wide distribution of avidin in avian, reptilian and amphibian species could be explained on the basis of its vital functions such as antimicrobial or antifungal, metabolic and immunomodulatory actions. The ontogeny of the progestin-dependent avidin synthesis is a complex event involving oviductal differentiation by steroid hormones leading to a specific gene expression. The first phase in oviductal differentiation by estrogens is characterized by a new chromatin organization and by an infiltration of progesterone receptor (PR)-containing mesenchymal cells into the subepithelial mucosa leading to epithelial cell differentiation ("mesenchymal and epithelial cell interaction"). The second phase in the differentiation of progestin-induced response is dependent on the presence of PR in the secretory cells. Two kinds of PR expression occur in the oviduct. The first is a "constitutive PR" and is found in the epithelial, submucosal and peritoneal cells of the immature chick oviduct without steroid treatment, and the second is an "inducible PR" found especially in the mucosal mesenchymal and smooth muscle cells. Avidin production requires PR in the target cells, but not all PR-containing cells can produce avidin. Therefore, in addition to PR, other transcription factors are needed to define the target cell specificity of the response to progestins. Earlier biochemical studies suggested that cytosolic and/or nuclear unoccupied PR was complexed as an 8 S form with the heat shock protein 90 (hsp90). Our immunohistochemical results, however, indicate that PR in vivo is not bound to hsp90, which is located entirely in the cytoplasm, whereas PR is an entirely nuclear protein in both ligand-occupied and unoccupied forms. Therefore, we assume that PR is a monomeric (4S) or homodimeric (5S) (chromatin?) protein associated to DNA. Ligand binding to PR appears to lead to a conformational change, dimer formation, tighter binding to PRE (progesterone responsive element) and to transcription factors, phosphorylation and proteolysis of PR as well as a chromatin change.(ABSTRACT TRUNCATED AT 400 WORDS)

Enhanced peroxisomal beta-oxidation of fatty acids and glutathione metabolism in rats exposed to phenoxyacetic acids.

Peroxisomal beta-oxidation of fatty acids and the activities of glutathione-metabolizing enzymes in rat liver were measured after administration of 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxyacetic acid (MCPA), clofibrate [ethyl], 2-(p-chlorophenoxy)-2-methylpropionate], glyphosate (N-phosphonomethyl glycine, a herbicide not structurally related to phenoxy acids) or saline for 14 days. beta-Oxidation increased by 6-fold in the group given clofibrate, 3-fold in the 2,4-D-treated group, and 2-fold in the MCPA-treated group over the level in the controls (saline-treated). Glyphosate did not increase beta-oxidation. No significant change in reduced glutathione content from that in controls was found in any of the treated groups. Glutathione reductase activity increased by about 40% after administration of either 2,4-D or MCPA, and glutathione peroxidase activity increased by 30% in animals given MCPA. A slight decrease in glutathione S-transferase activity was found in the group treated with clofibrate. The marked increases in peroxisomal beta-oxidation of fatty acids were accompanied by only minor changes in the activities of enzymes involved in glutathione-dependent inactivation of organic hydroperoxides and other oxygen-centred reactive agents.